DESCRIPTION (Verbatim from applicant's abstract): This is a medicinal chemistry proposal designed to develop new methods for inhibiting an important class of protease, those that are allosterically activated by substrate. Inhibitors of the botulinum toxin metalloproteases will be designed and synthesized based on two approaches: 1. design and synthesis of peptidyl transition-state analog inhibitors; and 2. Design based on the geometry of the zinc binding domain in the X-ray crystal structure of Botulinum toxin A. Botulinum toxin (BoNT) and tetanus toxin (TeNT) are converted in vivo to zinc metalloproteases which cleave single amide bonds in VAMP/synaptobrevin, SNAP25 and syntaxin. These metallo-proteases are the most selective proteases yet identified. Structure-activity data obtained with peptide substrates have established that substrate converts the BoNT metalloproteases from an inactive form to an active form. Examination of the 3.1 A X-ray crystal structure of native BoNT/A toxin provides insights into this unique substrate selectivity and also suggests how novel structures can be designed that will bind tightly to the INACTIVE protease, thereby preventing proteolysis. We postulate that it should be possible to stabilize the inactive form of the various BoNT metalloproteases; these inhibitors would be expected to have unique specificities and should not inhibit endogenous mammalian proteases. This new approach to the design of novel protease inhibitors will be applicable to other substrate activated proteases, many of which function in human biology and are implicated in disease.